Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease resulting from the loss of upper and lower motor neurons, typically fatal within 5 years of symptom onset. Our studies during the initial funding period have discovered protein biomarkers in ALS and candidate diagnostic biomarkers for ALS. However ALS progresses much more rapidly in some patients (death within 1 year from diagnosis) while others progress much more slowly (greater than 10 years from diagnosis to death). It is unclear why patients progress at quite differing rates, and few studies have explored disease progression within individual patients over time. We propose in this next funding period to generate a novel resource of longitudinal blood and CSF samples collected from ALS patients, neurologic disease controls, and healthy controls. We have extensive experience in collecting longitudinal samples from patients and controls and already have preliminary data indicating specific protein levels change during ALS disease progression. We will use these longitudinal samples to explore proteomic and extracellular RNAs (exRNA) to discover biomarkers for ALS disease progression. We will use both targeted and unbiased proteomic technologies, as well as novel exRNA methodologies in both blood and CSF. We hypothesize that specific biomarker signatures can be identified that correlate to clinical parameters of disease progression (fast vs. slow), therefore providing insight into molecular mechanisms that regulate disease progression. Our proposed studies will provide novel insight into proteomic changes during disease progression and characterize exRNAs in blood and CSF to discover novel exRNA biomarkers of disease progression. Finally, we will combine these complex proteomic and genomic datasets to discover biomarker panels for ALS disease progression.